This laboratory has recently immortalized (using the SV 40 large T antigen) all the cell types contributing to a developing seminiferous tubule in the mouse testis. The availability of these cell lines represents a major breakthrough in our ability to study spermatogenesis in vitro, to identify testis-specific factors that control gene expression and to characterize the molecules that control gametogenesis. The specific aims of the parent grant are: I. To characterize the functionality, hormonal responsiveness and iodogenic potential, of the Sertoli, peritubular, and Leydig cells. II. To investigate the ability of the in vitro spermatogenic tubule to ort normal germ cell differentiation. III. To investigate the ability of our immortalized germ cell line to erentiate in vitro. IV. To immortalize primordial germ cells and spermatogonia following the above or improved immortalization strategies and investigate this ability to differentiate in vitro. The current FIRCA proposal will extend the reach of the parent grant by focusing on identifying molecules that are uniquely involved in the paracrine regulation of spermatogenesis. Accordingly, the specific aim of this FIRCA application is: V. To identify and clone molecules that may be involved in the paracrine lation of gametogenesis, using subtraction cDNA libraries derived our immortalized cell lines.